Reduced voltage-activated Ca2+ release flux in muscle fibers from a rat model of Duchenne dystrophy.

The potential pathogenic role of disturbed Ca2+ homeostasis in Duchenne muscular dystrophy (DMD) remains a complex, unsettled issue. We used muscle fibers isolated from 3-mo-old DMDmdx rats to further investigate the case. Most DMDmdx fibers exhibited no sign of trophic or morphology distinction as compared with WT fibers and mitochondria and t-tubule membrane networks also showed no stringent discrepancy.

Detection of Ca2+ transients near ryanodine receptors by targeting fluorescent Ca2+ sensors to the triad.

In intact muscle fibers, functional properties of ryanodine receptor (RYR)-mediated sarcoplasmic reticulum (SR) Ca2+ release triggered by activation of the voltage sensor CaV1.1 have so far essentially been addressed with diffusible Ca2+-sensitive dyes. Here, we used a domain (T306) of the protein triadin to target the Ca2+-sensitive probe GCaMP6f to the junctional SR membrane, in the immediate vicinity of RYR channels, within the triad region.

Effects of maternal protein restriction on central and peripheral renin-angiotensin systems in male rat offspring.

Aims: We investigated the involvement of the renin angiotensin system (RAS) on the cardiorespiratory control in rats from dams fed with a low-protein diet.

Main Methods: Male offspring were obtained from dams fed a normoprotein diet (NP, 17% casein) and low-protein diet (LP, 8% casein) during pregnancy and lactation. Direct measurements of arterial pressure (AP), heart rate (HR) and respiratory frequency (RF) were recorded in awake 90-day-old at resting and after losartan potassium through either intracerebroventricular (ICV) microinjections or intravenous (IV) administration.

Treatment with the essential amino acid L-tryptophan reduces masticatory impairments in experimental cerebral palsy.

Children with cerebral palsy (CP) often exhibit difficulties in feeding resulting from deficits in chewing. This study investigates the therapeutic potential of L-tryptophan (TRI) to reduce deficits in chewing in rats subjected to an experimental model of CP. A total of 80 Wistar albino rats were used.

Twice-a-day training improves mitochondrial efficiency, but not mitochondrial biogenesis, compared with once-daily training.

Exercise training performed with lowered muscle glycogen stores can amplify adaptations related to oxidative metabolism, but it is not known if this is affected by the "train-low" strategy used (i.e., once-daily versus twice-a-day training).

Differential effects of maternal high-fat/high-caloric or isocaloric diet on offspring's skeletal muscle phenotype.

Unlabelled: Aim This study sought to investigate the effects of two different maternal high-fat diets, during gestation and lactation, on the morphology of the skeletal muscle of the adult offspring rats.

Methods: Female Wistar rats were fed Control (C) or High-fat/high-caloric (HH) or High-fat/isocaloric (HI) diet during gestation and lactation. The somatic growth of the offspring was measured throughout lactation.

Carotid body removal normalizes arterial blood pressure and respiratory frequency in offspring of protein-restricted mothers.

The aim of this study is to evaluate the short-term and long-term effects elicited by carotid body removal (CBR) on ventilatory function and the development of hypertension in the offspring of malnourished rats. Wistar rats were fed a normo-protein (NP, 17% casein) or low-protein (LP, 8% casein) diet during pregnancy and lactation. At 29 days of age, the animals were submitted to CBR or a sham surgery, according to the following groups: NP-cbr, LP-cbr, NP-sham, or LP-sham.

Impaired excitation-contraction coupling in muscle fibres from the dynamin2 mouse model of centronuclear myopathy.

Key Points: Dynamin 2 is a ubiquitously expressed protein involved in membrane trafficking processes. Mutations in the gene encoding dynamin 2 are responsible for a congenital myopathy associated with centrally located nuclei in the muscle fibres. Using muscle fibres from a mouse model of the most common mutation responsible for this disease in humans, we tested whether altered Ca signalling and excitation-contraction coupling contribute to muscle weakness.

Phosphatidylinositol 3-kinase inhibition restores Ca2+ release defects and prolongs survival in myotubularin-deficient mice.

Mutations in the gene encoding the phosphoinositide 3-phosphatase myotubularin (MTM1) are responsible for a pediatric disease of skeletal muscle named myotubular myopathy (XLMTM). Muscle fibers from MTM1-deficient mice present defects in excitation-contraction (EC) coupling likely responsible for the disease-associated fatal muscle weakness. However, the mechanism leading to EC coupling failure remains unclear.

Involvement of Cyclophilin D and Calcium in Isoflurane-induced Preconditioning.

Background: The mitochondrial permeability transition pore (PTP) has been established as an important mediator of ischemia-reperfusion-induced cell death. The matrix protein cyclophilin D (CypD) is the best known regulator of PTP opening. Therefore, the authors hypothesized that isoflurane, by inhibiting the respiratory chain complex I, another regulator of PTP, might reinforce the myocardial protection afforded by CypD inhibition.

Synergistic protective effect of cyclosporin A and rotenone against hypoxia-reoxygenation in cardiomyocytes.

Reperfusion of the heart after an ischemic event leads to the opening of a nonspecific pore in the inner mitochondrial membrane, the mitochondrial permeability transition pore (mPTP). Inhibition of mPTP opening is an effective strategy to prevent cardiomyocyte death. The matrix protein cyclophilin-D (CypD) is the best-known regulator of mPTP opening.

Analysis of the capture and influence of nanoparticles on the cytotoxic effects of 7-ketocholesterol on cardiac cells: investigation by flow cytometry and by dynamic and spectral imaging microscopy associated with factor analysis of medical image sequences.

Objective: To evaluate the capture of nanoparticles (quantum dots [QDs], fluorospheres) by nonbeating mouse cardiac cells (HL1-NB) cultured without or with 7-ketocholesterol (7KC) found at an increased level in the plasma of atherosclerotic patients and to simultaneously analyze their cytotoxic, proinflammatory and oxidative properties.

Study Design: Flow cytometry (FCM), confocal laser scanning microscopy and subsequent factor analysis image processing were used to characterize the uptake of nanoparticles and to define their cytotoxicity, evaluated by enhanced permeability to SYTOX Green, release of lactate dehydrogenase (LDH) and morphologic nuclear changes determined with Hoechst 33342. Proinflammatory effects were estimated by enzyme linked immunoassay to quantify IL-8 and MCP-1 secretion.

Iron nanoparticles increase 7-ketocholesterol-induced cell death, inflammation, and oxidation on murine cardiac HL1-NB cells.

Objective: To evaluate the cytotoxicity of iron nanoparticles on cardiac cells and to determine whether they can modulate the biological activity of 7-ketocholesterol (7KC) involved in the development of cardiovascular diseases. Nanoparticles of iron labeled with Texas Red are introduced in cultures of nonbeating mouse cardiac cells (HL1-NB) with or without 7-ketocholesterol 7KC, and their ability to induce cell death, pro-inflammatory and oxidative effects are analyzed simultaneously.

Study Design: Flow cytometry (FCM), confocal laser scanning microscopy (CLSM), and subsequent factor analysis image processing (FAMIS) are used to characterize the action of iron nanoparticles and to define their cytotoxicity which is evaluated by enhanced permeability to SYTOX Green, and release of lactate deshydrogenase (LDH).

High-resolution tomographic diffractive microscopy of biological samples.

The authors have developed a tomographic diffractive microscope that combines microholography with illumination from an angular synthetic aperture. It images specimens relative to their complex index of refraction distribution (index and absorption) and permits imaging of unlabelled specimens, with high lateral resolution. The authors now study its use for biological applications, and imaged several preparations with fluorescence confocal microscopy and tomographic diffractive microscopy.

Mitochondrial dynamics in heart cells: very low amplitude high frequency fluctuations in adult cardiomyocytes and flow motion in non beating Hl-1 cells.

The arrangement and movement of mitochondria were quantitatively studied in adult rat cardiomyocytes and in cultured continuously dividing non beating (NB) HL-1 cells with differentiated cardiac phenotype. Mitochondria were stained with MitoTracker Green and studied by fluorescent confocal microscopy. High speed scanning (one image every 400 ms) revealed very rapid fluctuation of positions of fluorescence centers of mitochondria in adult cardiomyocytes.

Comparative analysis of the bioenergetics of adult cardiomyocytes and nonbeating HL-1 cells: respiratory chain activities, glycolytic enzyme profiles, and metabolic fluxes.

Comparative analysis of the bioenergetic parameters of adult rat cardiomyocytes (CM) and HL-1 cells with very different structure but similar cardiac phenotype was carried out with the aim of revealing the importance of the cell structure for regulation of its energy fluxes. Confocal microscopic analysis showed very different mitochondrial arrangement in these cells. The cytochrome content per milligram of cell protein was decreased in HL-1 cells by a factor of 7 compared with CM.

Ciliary beating recovery in deficient human airway epithelial cells after lentivirus ex vivo gene therapy.

Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia.

Telomere uncapping during in vitro T-lymphocyte senescence.

Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length.

Structure-function relationships in the regulation of energy transfer between mitochondria and ATPases in cardiac cells.

The present study discusses the role of structural organization of cardiac cells in determining the mechanisms of regulation of oxidative phosphorylation and interaction between mitochondria and ATPases. In permeabilized adult cardiomyocytes, the apparent K(m) (Michaelis-Menten constant) for ADP in the regulation of respiration is far higher than in mitochondria isolated from the myocardium. Respiration of mitochondria in permeabilized cardiomyocytes is effectively activated by endogenous ADP produced by ATPases from exogenous ATP, and the activation of respiration is associated with a decrease in the apparent K(m) for ATP in the regulation of ATPase activity compared with this parameter in the absence of oxidative phosphorylation.

New generation photon-counting cameras: algol and CPNG.

Algol and Comptage de Photons Nouvelle Génération (CPNG) are new generation photon counting cameras developed for high angular resolution in the visible by means of optical aperture synthesis and speckle interferometry and for photon noise limited fast imaging of biological targets. They are intensified CCDs. They have been built to benefit from improvements in photonic commercial components, sensitivity, and personal computer workstations processing power.

Effects of caspase inhibitors (z-VAD-fmk, z-VDVAD-fmk) on Nile Red fluorescence pattern in 7-ketocholesterol-treated cells: investigation by flow cytometry and spectral imaging microscopy.

Background: The 7-ketocholesterol (7KC)-induced cell death has some characteristics of apoptosis and is associated with polar lipid accumulation. So, we investigated the effects of the broad-spectrum caspase inhibitor z-VAD-fmk and of the caspase-2 inhibitor z-VDVAD-fmk on lipid profile evaluated by staining with Nile Red (NR).

Methods: The 7KC-treated human monocytic U937 cells were cultured in the absence or in the presence of the caspase inhibitors z-VAD-fmk or z-VDVAD-fmk.

Flow cytometry and spectral imaging multiphoton microscopy analysis of CD36 expression with quantum dots 605 of untreated and 7-ketocholesterol-treated human monocytic cells.

Objective: To evaluate CD36 expression with quantum dots 605 (QDs 605) on untreated and 7-ketocholesterol (7KC)-treated monocytic U937 cells by flow cytometry (FCM) and confocal and multiphoton laser scanning microscopy (CLSM).

Study Design: Cells were analyzed by CLSM, following flow cytometric quantification of CD36 expression and 7KC uptake. Image sequences were obtained by spectral analysis in monophoton and multiphoton CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm to differentiate emission spectra.

Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells. Importance of cell structure/organization for respiration regulation.

The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25+/-4 microM) and seven times lower in normally cultured HL-1 cells (47+/-15 microM) than in permeabilized primary cardiomyocytes (360+/-51 microM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration.

Confocal laser scanning microscopy using dialkylcarbocyanine dyes for cell tracing in hard and soft biomaterials.

The aim of this work was to study, in vitro, cell colonization of two biomaterials currently used for bone and cartilage repair, this step being important to understand the function of engineered tissues. Current methods that use histological approaches are not always suited to tissue-engineering analysis. We, therefore, set up a protocol to assess cell distribution, utilizing noninvasive confocal microscopy and fluorescent labels with a far red emission wavelength to optimize scaffold transparency and minimize light scattering.

Analysis of CD36 expression on human monocytic cells and atherosclerotic tissue sections with quantum dots: investigation by flow cytometry and spectral imaging microscopy.

Objective: To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM).

Study Design: U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes.